Recombinant proteins are proteins that have been created by combining the DNA of different organisms. This process often involves taking a gene from one organism, inserting it into an empty “vector” in another organism’s DNA, and then letting the vector insert itself back into the first organism’s DNA.
Recombinant protein production is important for medicine, biotechnology research, food science, biofuel development, just about any field involving life sciences. In this article, you will learn about recombinant proteins.
To create recombinant proteins, you first need to break open your cells. The process of breaking open the cell is called lysis which can be achieved by using a detergent or physical stress like shear force via pressure or sound waves.
The goal here is not only to release the DNA into a solution but also to separate it from everything else in the cellular environment so that it will work for your experiments without interference or contamination. For example, if you were studying protein-protein interactions between two different genes inside one bacteria’s chromosome, then contaminating another gene with this experiment might cause problems because they are interacting with each other already, and adding additional “noise” could skew the data.
Lysis – How To:
There are many different ways to lyse cells. Standard methods include adding a detergent-like sodium dodecyl sulfate (SDS) or Nonidet P-40, which will break open the cell membranes by disrupting hydrogen bonds between proteins and lipids in the membrane. You can also use sound waves via ultrasonic treatment. A third option is by mechanically shearing the cells with grinders or presses.
Alternative Method For Lysis: Use Magnesium Chloride Solution
Detergents are commonly used to lyse cells, but another method doesn’t use them: Using magnesium chloride solution. The salt causes osmosis, where water migrates into the cell until it bursts.
After you have lysed your cells, you need to separate the protein of interest. This process is called solubilization, and it often involves adding other chemicals like urea which helps with heat stability, or guanidine hydrochloride (GdnHCl), which will denature proteins so that they are in their unfolded state. This makes them easier to work with because now they can interact with the detergent.
Solubilization – How To:
There are a few ways to keep the protein from clumping up when we add these harsh chemicals:
- i) Using an anti-clotting agent such as ethylenediaminetetraacetic acid (EDTA)
- ii) Adjusting pH levels
iii)Changing ionic strength by adding salts/salts like ammonium sulfate
After you have solubilized your protein, the third step is purification. Many different types of chromatography can be used to separate molecules based on their properties or characteristics, including size, charge, and affinity for other chemicals. Two common methods include ion-exchange chromatography – where charged particles bind to resins depending on their opposite charges/affinity – and gel filtration, which separates proteins by molecular weight(how big they are).
Chromatography – How To:
As mentioned previously, many different forms of chromatography can be used to purify proteins after solubilization, including ion-exchange chromatography; gel filtration columns; high-performance liquid chromatography (HPLC); size-exclusion HPLC column, which utilizes a silica gel or agarose matrix to separate molecules based on their size, charge, and affinity for other chemicals. Each one separates the protein by its molecular weight.
Dialysis is the process of filtering out salt, detergents, or other molecules that might affect how your protein folds with a semi-permeable membrane. This allows you to keep only small proteins below 50 kDa. The pore size in the membrane is important when choosing which one to use because they are rated by their molecular weight cutoff (MWCO).
Dialysis – How To:
Common ways include using dialysis tubing/bags, columns made up of membranes where different sized pores allow for separation based on molecular weight, depending on the kind of molecule in question and its MWCO rating. You can choose the most suitable from the many types of membranes available today, including cellulose acetate membranes, regenerated cellulose membranes, polyethersulfone membranes, and chromatic membrane filters.
In summary, recombinant protein purification can be done in three simple steps. First, you need to lysate the cells; second, solubilize your proteins with whatever chemicals you want depending on what kind of molecule is in question and its MWCO rating; thirdly, separate the protein by using chromatography or dialysis methods that will allow you to keep only proteins below a certain molecular weight cutoff.